Inhibitors of HIV protease useful for the treatment of AIDS

ABSTRACT

The present invention provides novel HIV protease inhibitors, pharmaceutical formulations containing those compounds and methods of treating and/or preventing HIV infection and/or AIDS.

BACKGROUND OF THE INVENTION

A retrovirus designated human immuno-deficiency virus (HIV) is thecausative agent of the complex disease termed Acquired Immune DeficiencySyndrome (AIDS), and is a member of the lentivirus family ofretroviruses. M. A. Gonda, F. Wong-Staal, R. C. Gallo, "SequenceHomology and Morphological Similarity of HTLV III And Visna Virus, APathogenic Lentivirus", Science, 227, 173, (1985); P. Sonigo, N. Alizon,et al., "Nucleotide Sequence of the Visna Lentivirus: Relationship tothe AIDS Virus", Cell, 42, 369, (1985). The complex disease AIDSincludes progressive destruction of the immune system and degenerationof the central and peripheral nervous systems. The HIV virus waspreviously known or referred to as LAV, HTLV-III or ARV.

A common feature of retrovirus replication is the post-translationalprocessing of precursor polyproteins by a virally encoded protease togenerate mature vital proteins required for viral assembly and function.Interruption of this processing appears to prevent the production ofnormally infectious virus. Unprocessed structural proteins also havebeen observed in clones of non-infectious HIV strains isolated fromhuman patients. The results suggest that the inhibition of HIV proteaserepresents a viable method for the treatment or prevention of AIDS andthe treatment or prevention of infection by HIV.

The HIV genome encodes structural protein precursors known as gag andpol, which are processed to afford the protease, reverse transcriptaseand endonuclease/integrase. The protease further cleaves gag and gag-polpolyproteins to yield mature structural proteins of the virus core.

Considerable efforts are being directed toward the control of HIV bymeans of the structural protein precursors which are processed to yieldthe retroviral protease, reverse transcriptase andendonuclease/integrase. For example, the currently used therapeutic,AZT, is an inhibitor of the viral reverse transcriptase. H. Mitsuya, N.S. Broder, "Inhibition of the In Vitro Infectivity in Cytopathic Effectsof HTLV III", Proc. Natl. Acad. Sci. USA, 83, 1911 (1986).

Research efforts have also been directed toward HIV protease inhibitors.For example, Europpean Patent Application (EPA) 361 341; EPA 346 847;EPA 402 646; and EPA 337 714 all disclose compounds which are said to beuseful as HIV protease inhibitors.

Unfortunately, many of the known compounds suffer from toxicityproblems, lack of bioavailability or short in vivo half-lives. Thus,despite the recognized therapeutic potential associated with a proteaseinhibitor and the research efforts expended thus far, a viabletherapeutic agent has not yet emerged.

Accordingly, a primary object of the present invention is to providenovel protease inhibitors which are useful in the treatment orprevention of HIV infection and/or the resulting acquired immunedeficiency syndrome (AIDS).

A further object of the present invention is to provide therapeuticcompositions that are of value in the treatment or prevention of HIVinfection and/or AIDS.

Still another object is to provide methods for the treatment orprevention of HIV infection and/or AIDS.

Other objects, features and advantages will become apparent to thoseskilled in the art from the following description and claims.

SUMMARY OF THE INVENTION

The present invention relates to compounds of formula I, below, andpharmaceutically acceptable salts thereof that inhibit the proteaseencoded by human immunodeficiency virus (HIV) type 1 (HIV-1) and type 2(HIV-2). These compounds are useful in the treatment or prevention ofinfection by HIV and the treatment or prevention of the resultingacquired immune deficiency syndrome (AIDS). The compounds, theirpharmaceutically acceptable salts, and the pharmaceutical compositionscan be used alone or in combination with other antivitals,immunomodulators, antibiotics or vaccines. Methods of treating orpreventing AIDS, methods of treating or preventing HIV infection andmethods of inhibiting HIV replication are disclosed.

The present invention relates to a method of inhibiting HIV protease inan HIV infected cell, a cell susceptible to HIV infection or a primatein need thereof, thus treating or preventing HIV infection and/or AIDS,comprising administering an effective amount of a compound of formula I##STR1## wherein:

R is hydrogen, formyl or C₂ -C₆ alkanoyl;

q is 0, 1 or 2;

R¹ is aryl or C₅ -C₇ cycloalkyl;

R² is an amino acid side chain, --CH₂ --R^(2a), --CH₂--C(O)--NH--A--R^(2a) or --CH₂ --C(O)--OR^(2a), where

A is a bond, --(CH₂)_(v) --, --(CH₂)_(m) --O--(CH₂)_(n) -- or --(CH₂)_(m) NR⁰ --(CH₂)_(n) --, where

m and n are independently 0, 1 or 2;

v is 0, 1, 2 or 3;

R^(2a) is aryl, heterocycle or unsaturated heterocycle;

X is a group having the structure: ##STR2## Y is aryl or unsaturatedheterocycle; Y¹ is heterocycle;

R^(3a) is a group having the structure: ##STR3## R^(3b) is a grouphaving the structure: ##STR4## where: p is 4 or 5;

l is 3, 4 or 5;

R⁴ at each occurrence is independently hydrogen, C₁ -C₆ alkyl orhydroxy(C₁ -C₄)alkyl; and

R⁵ and R⁶ are independently selected from hydrogen, hydroxy, C₁ -C₆alkyl, C₁ -C₆ alkoxy, amino, C₁ -C₄ alkylamino, hydroxy(C₁ -C₄)alkyl,carboxy, C₁ -C₄ alkoxycarbonyl, carbamoyl, N--(C₁ -C₄)alkylcarbamoyl,aryl, heterocycle or unsaturated heterocycle; or a pharmaceuticallyacceptable salt thereof.

The present invention also relates to a compound of formula I, or apharmaceutically acceptable salt thereof, wherein A, R, R¹, R², q and Xare as defined above.

The present invention further provides pharmaceutical formulationscomprising a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, in combination with a pharmaceuticallyacceptable carrier, diluent or excipient therefor.

A further embodiment of the present invention is a class of novelintermediates useful for preparing the compounds of formula I. Theintermediates have the formula ##STR5## where:

R¹ and X are as defined above for formula I; or a pharmaceuticallyacceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new compounds of formula I, as describedabove, that are useful for treating or preventing HIV infection and/orAIDS.

All temperatures stated herein are in degrees Celsius (°C.). All unitsof measurement employed herein are in weight units except for liquidswhich are in volume units.

As used herein, the term "C₁ -C₆ alkyl" represents a straight orbranched alkyl chain having from one to six carbon atoms. Typical C₁ -C₆alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, t-butyl, pentyl, neo-pentyl, hexyl and the like. The term "C₁-C₆ alkyl" includes within its definition the term "C₁ -C₄ alkyl".

"Halo" represents chloro, fluoro, bromo or iodo.

"Halo(C₁ -C₄)alkyl" represents a straight or branched alkyl chain havingfrom one to four carbon atoms with 1-3 halogen atoms attached to it.Typical halo(C_(1-C) ₄)alkyl groups include chloromethyl, 2-bromoethyl,1-chloroisopropyl, 3-fluoropropyl, 2,3-dibromobutyl, 3-chloroisobutyl,iodo-8-butyl, trifluoromethyl and the like.

"Cyano(C₁ -C₄)alkyl" represents a straight or branched alkyl chainhaving from one to four carbon atoms with a cyano group attached to it.Typical cyano(C₁ -C₄)-alkyl groups include cyanomethyl, 2-cyanoethyl,3-cyanopropyl, 2-cyanoisopropyl, 4-cyanobutyl and the like.

"C₁ -C₄ alkylthio" represents a straight or branched alkyl chain havingfrom one to four carbon atoms attached to a sulfur atom. Typical C₁ -C₄alkylthio groups include methylthio, ethylthio, propylthio,isopropylthio, butylthio and the like.

"C₁ -C₄ alkylthio(C₁ -C₄)alkyl" represents a straight or branched alkylchain containing from one to four carbon atoms with a C₁ -C₄ alkylthiogroup attached to it. Typical C₁ -C₄ alkylthio(C₁ -C₄)alkyl groupsinclude methylthioethyl, ethylthiobutyl, propylthioisopropyl,isopropylthiomethyl, butylthioethyl and the like.

"C₁ -C₄ alkylamino" represents a straight or branched alkylamino chainhaving from one to four carbon atoms attached to an amino group. TypicalC₁ -C₄ alkylamino groups include methylamino, ethylamino, propylamino,isopropylamino, butylamino, sec-butylamino and the like.

"Di(C₁ -C₄)alkylamino" represents a straight or branched dialkylaminochain having two alkyl chains of from one to four carbon atoms attachedto a common amino group. Typical di(C₁ -C₄)alkylamino groups includedimethylamino, ethylmethylamino, methylisopropylamino,8-butylisopropyl-amino, di-t-butylamino and the like.

"C₁ -C₄ alkoxy" represents a straight or branched alkyl chain havingfrom one to four carbon atoms attached to an oxygen atom. Typical C₁ -C₄alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy andthe like.

"C₁ -C₄ alkoxycarbonyl" represents a straight or branched alkoxy chainhaving from one to four carbon atoms attached to a carbonyl moiety.Typical C₁ -C₄ alkoxy-carbonyl groups include methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,t-butoxycarbonyl and the like.

"C₂ -C₆ alkanoyl" represents a straight or branched alkyl chain havingfrom one to five carbon atoms attached to a carbonyl moiety. Typical C₂-C₆ alkanoyl groups include ethanoyl, propanoyl, isopropanoyl, butanoyl,t-butanoyl, pentanoyl, hexanoyl, 3-methylpentanoyl and the like.

"Carbamoyl(C₁ -C₄)alkyl" represents a straight or branched alkyl chainhaving from one to four carbon atoms with a carbamoyl group attached toit. Typical carbamoyl(C₁ -C₄)alkyl groups include carbamoylmethyl,carbamoylethyl, carbamoylpropyl, carbamoylisopropyl, carbamoylbutyl andcarbamoyl-t-butyl and the like.

"C₅ -C₇ cycloalkyl" represents a saturated hydrocarbon ring structurecontaining from five to seven carbon atoms which is unsubstituted orsubstituted with 1, 2 or 3 substituents independently selected fromhalo, halo(C₁ -C₄)alkyl, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, carboxy, C₁ -C₄alkoxycarbonyl, carbamoyl, C₁ -C₄ alkylcarbamoyl, amino, C₁ -C₄alkylamino, di(C₁ -C₄)alkylamino or a group having the structure--(CH₂)_(a) --R⁷ where a is 1, 2, 3 or 4 and R⁷ is hydroxy, C₁ -C₄alkoxy, carboxy, C₁ -C₄ alkoxycarbonyl, amino, carbamoyl, C₁ -C₄alkylamino or di(C₁ -C₄ ) alkylamino. Typical C₅ -C₇ cycloalkyl groupsinclude cyclopentyl, cyclohexyl, cycloheptyl, 3-methylcyclopentyl,4-ethoxycyclohexyl, 5-carboxycycloheptyl, 6-chlorocyclohexyl and thelike.

The term "heterocycle" represents an unsubstituted or substituted stable5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclicring which is saturated and which consists of carbon atoms and from oneto three heteroatoms selected from the group consisting of nitrogen,oxygen or sulfur, and wherein the nitrogen and sulfur heteroatoms mayoptionally be oxidized, and the nitrogen heteroatom may optionally bequaternized and including a bicyclic group in which any of theabove-defined heterocyclic rings is fused to a benzene ring. Theheterocyclic ring may be attached at any heteroatom or carbon atom whichaffords a stable structure. The heterocycle is unsubstituted orsubstituted with 1, 2 or 3 substituents independently selected fromhalo, halo(C_(1-C) ₄)alkyl, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, carboxy, C₁ -C₄alkoxycarbonyl, carbamoyl, C₁ -C₄ alkylcarbamoyl, amino, C_(1-C) ₄alkylamino, di(C₁ -C₄)alkylamino or a group having the structure--(CH₂)_(a) --R⁷ where a is 1, 2, 3 or 4; and R⁷ is hydroxy, C₁ -C₄alkoxy, carboxy, C₁ -C₄ alkoxycarbonyl, amino, carbamoyl, C.sub. 1 -C₄alkylamino or di(C₁ -C₄)alkylamino.

The term "unsaturated heterocycle" represents an unsubstituted orsubstituted stable 5- to 7-membered monocyclic or 7- to 10-memberedbicyclic heterocyclic ring which has one or more double bonds and whichconsists of carbon atoms and from one to three heteroatoms selected fromthe group consisting of nitrogen, oxygen or sulfur, and wherein thenitrogen and sulfur heteroatoms may optionally be oxidized, and thenitrogen heteroatom may optionally be quarternized and including abicyclic group in which any of the above-defined heterocyclic rings isfused to a benzene ring. The unsaturated heterocyclic ring may beattached at any heteroatom or carbon atom which affords a stablestructure. The unsaturated heterocycle is unsubstituted or substitutedwith 1, 2 or 3 substituents independently selected from halo, halo(C₁-C₄)alkyl, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, carboxy, C₁ -C₄ alkoxycarbonyl,carbamoyl, C₁ -C₄ alkylcarbamoyl, amino, C₁ -C₄ alkylamino, di(C₁-C₄)alkylamino or a group having the structure --(CH₂ )_(a) --R⁷ where ais 1, 2, 3 or 4; and R⁷ is hydroxy, C₁ -C₄ alkoxy, carboxy, C₁ -C₄alkoxycarbonyl, amino, carbamoyl, C₁ -C₄ alkylamino or di (C₁ -C₄)alkylamino.

Examples of such heterocycles and unsaturated heterocycles includepiperidinyl, piperazinyl, azepinyl, pyrrolyl, 4-piperidonyl,pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl,quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazolyl, benzopyranyl,benzothiazolyl, benzoazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl,thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl-sulfoxide,thiamorpholinylsulfone, oxadiazolyl, triazolyl, tetrahydroquinolinyl,tetrahydrisoquinolinyl, 3 -methyl-imidazolyl, 3-methoxypyridyl,4-chloroquinolinyl, 4-aminothiazolyl, 8 -methylquinolinyl, 6-chloroquinoxalinyl, 3-ethylpyridyl, 6-methoxybenzimidazolyl,4-hydroxyfuryl 4-methylisoquinolinyl, 6,8-dibromoquinolinyl,4,8-dimethylnaphthyl, 2-methyl-1,2,3,4-tetrahydroisoquinolinyl,N-methylquinolin-2-yl, 2-8-butoxycarbonyl-1,2,3,4-isoquinotin-7-yl andthe like.

"Aryl" represents a phenyl or naphthyl ring which is optionallysubstituted with 1, 2 or 3 substituents independently selected fromhalo, morpholino (C₁ -C₄) alkoxycarbonyl, pyridyl(C₁ -C₄)alkoxycarbonyl,C₁ -C₄ alkyl, C₁ -C₄ alkoxy, carboxy, C₁ -C₄ alkoxycarbonyl, carbamoyl,carbamoyl(C₁ -C₄)alkyl, amino, C₁ -C₄ alkylamino, di(C₁ -C₄)-alkylaminoor a group of the formula --(CH₂)_(a) --R⁷ where a is 1, 2, 3 or 4; andR⁷ is hydroxy, C₁ -C₄ alkoxy, carboxy, C₁ -C₄ alkoxycarbonyl, amino,carbamoyl, C₁ -C₄ alkylamino or di(C₁ -C₄)alkylamino. Typical arylgroups include 4-methylphenyl, 3-ethylnaphthyl, 2,5-dimethylphenyl,8-chloronaphthyl, 3-aminonaphthyl, 4-carboxyphenyl and the like.

The term "amino acid side chain" represents the distinctive atom orgroup bonded to an G-carbon atom also having bonded thereto a carboxylgroup and an amino group. These side chains are selected from thosefound on the following amino acids:

    ______________________________________                                               Alanine         Ala                                                           Arginine        Arg                                                           Asparagine      Asn                                                           Aspartic acid   Asp                                                           Cysteine        Cys                                                           Glutamine       Gln                                                           Glutamic acid   Glu                                                           Glycine         Gly                                                           Histidine       His                                                           Isoleucine      Ile                                                           Leucine         Leu                                                           Lysine          Lys                                                           Methionine      Met                                                           Phenylalanine   Phe                                                           Proline         Pro                                                           Serine          Ser                                                           Threonine       Thr                                                           Tryptophan      Trp                                                           Tyrosine        Tyr                                                           Valine          Val                                                    ______________________________________                                    

The term "amino-protecting group" as used in the specification refers tosubstituents of the amino group commonly employed to block or protectthe amino functionality while reacting other functional groups on thecompound. Examples of such amino-protecting groups include formyl,trityl, phthalimido, trichloroacetyl, chloroacetyl, bromoacety1,iodoacetyl or urethane-type blocking groups such as benzyloxycarbonyl,4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxy-carbonyl,2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl,1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl,2-(p-toluyl)prop-2-yloxl/carbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl,1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyl-oxycarbonyl,2-(4-toluylsulfonyl) ethoxycarbonyl, 2-(methylsulfonyl) ethoxycarbonyl,2- (triphenylphosphino)ethoxycarbonyl, fluorenylmethoxycarbonyl ( "FHOC") , 2-(trimethylsilyl) ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, 4-(decyloxy)benzyloxycarbonyl,isobornyloxycarbonyl, 1-piperidyloxycarbonyl and the like; orbenzoylmethylsulfonyl, 2-nitrophenylsulfenyl, diphenylphosphine oxideand like amino-protecting groups. The species of amino-protecting groupemployed is not critical so long as the derivatized amino group isstable to the condition of subsequent reaction(s) on other positions ofthe intermediate molecule and can be selectively removed at theappropriate point without disrupting the remainder of the moleculeincluding any other amino-protecting group (s) . Preferredamino-protecting groups are t-butoxycarbonyl (t-Boc) andbenzyloxycarbonyl (Cbz). Further examples of groups referred to by theabove terms are described by J. W. Barton, "Protective Groups in OrganicChemistry", J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973,Chapter 2, and T. W. Greene, "Protective Groups in Organic Synthesis",John Wiley and sons, New York, N.Y., 1981, Chapter 7.

The term "carboxy-protecting group" as used in the specification refersto substituents of the carboxy group commonly employed to block orprotect the carboxy functionality while reacting other functional groupson the compound. Examples of such carboxy-protecting groups includemethyl, p-nitrobenzyl, p-methylbenzyl, p-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylene-dioxybenzyl,benzhydryl, 4,4'-dimethoxybenzhydryl, 2,2',4,4'-tetramethoxy-benzhydryl,t-butyl, 8-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl,4,4',4"-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl,t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl,β-(di(butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)prop-1-en-3-yl and like moleties. A preferredcarboxy-protecting group is benzhydryl. Further examples of these groupsare found in E. Haslam, "Protective Groups in Organic Chemistry", J. G.W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W.Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons,New York, N.Y., 1981, Chapter 5.

The compounds of the present invention may have three asymmetric centersas denoted by the asterisks in the formula below. ##STR6##

As a consequence of these asymmetric centers, the compounds of thepresent invention can occur as mixtures of diastereomers, racemicmixtures and as individual enantiomers. All asymmetric forms, individualisomers and combinations thereof, are within the scope of the presentinvention.

As mentioned above, the invention includes the pharmaceuticallyacceptable salts of the compounds defined by formula I. Althoughgenerally neutral, a compound of this invention can possess asufficiently acidic, a sufficiently basic, or both functional groups,and accordingly react with any of a number of inorganic bases, andinorganic and organic acids, to form a pharmaceutically acceptable salt.

The term "pharmaceutically acceptable salt" as used herein, refers tosalts of the compounds of the above formula which are substantiallynon-toxic to living organisms. Typical pharmaceutically acceptable saltsinclude those salts prepared by reaction of the compounds of the presentinvention with a mineral or organic acid or an inorganic base. Suchsalts are known as acid addition and base addition salts.

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid and the like; and organic acids such asp-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like.

Examples of such pharmaceutically acceptable salts are the sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,y-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, napththalene-2-sulfonate,mandelate and the like. Preferred pharmaceutically acceptable acidaddition salts are those formed with mineral acids such as hydrochloricacid and hydrobromic acid, and those formed with organic acids such asmaleic acid and methanesulfonic acid.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, sodium carbonate, sodiumbicarbonate, potassium bicarbonate, calcium hydroxide, calciumcarbonate, and the like. The potassium and sodium salt forms areparticularly preferred.

It should be recognized that the particular counterion forming a part ofany salt of this invention is not of a critical nature, so long as thesalt as a whole is pharmacologically acceptable and as long as thecounterion does not contribute undesired qualities to the salt as awhole.

Preferred compounds of this invention are those compounds of formula Iwhere:

R is formyl or C₂ -C₆ alkanoyl;

A is a bond;

R¹ is aryl;

q is 0;

R² is --CH₂ --C (O)NH₂, --CH(CH₃)₂ or --CH₂ --C(O)--NH--A--R^(2a) ;##STR7##

R^(3a) is --C(O)--NR⁴ R⁴ where R⁴ is independently and at eachoccurrence hydrogen or C₁ -C₆ alkyl; or a pharmaceutically acceptablesalt thereof.

Of these compounds, more preferred compounds are those compounds offormula I where:

R is ethanoyl;

Y is phenyl;

Y¹ is decahydro-(4aS, 8aS)-isoquinolinyl;

R¹ is naphth-2-yl;

R² is --CH₂ --C(O)NH₂ ; and

R^(3a) is --C(O)--NH(t-butyl);

or a pharmaceutically acceptable salt thereof.

The most preferred compound is [2'R-(2'R*, 3'R*,6'S*)]--N(t-butyl)-2-[2'-hydroxy-3'-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"amino-1"-oxoethyl)]nonylbenzamide;

or a pharmaceutically acceptable salt thereof.

The following list of compounds is provided to further illustratecompounds of formula I included within the scope of the invention:

[2'R-(2'R*, 3'R*, 6'S*)]-N (8-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(1"methylethyl)]nonylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(C-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(imidazol-4"-ylmethyl)]nonylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(C-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylnaphthyl-2-carboxamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(8-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylquinoxalinyl -2-carboxamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylquinolinyl-2-carboxamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-amino]hexylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(methanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl -4'-aza-5',8'-dioxo-6"(2 "-amino-2 "-oxoethyl)-6-N(ethanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(propanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-naphth-2-ylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(butanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(methanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(ethanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(propanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*, 6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-N(butanoyl)amino]hexylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)-6-amino]hexylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(1"methylethyl)]nonylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(imidazol-4"-ylmethyl)]nonylbenzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3'-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(1"methylethyl)amino]hexyl benzamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3-phenylthiomethyl-4'-aza-5',8'-dioxo-6'-(imidazol-4"-ylmethyl)-6'-amino]hexylbenzamide;

[2'R-(2'R*, 3'R*,d'S*)]-N(8-butyl)-2-[2'-hydroxy-3'-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylnaphthyl-2-carboxamide;

[2'R-(2'R*, 3'R*,d'S*)]-N(8-butyl)-2-[2'-hydroxy-3'-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-hydroxy)]nonylnaphthyl-2-carboxamide;

[2'R-(2'R*, 3'R*,6'S*)]-N(8-butyl)-2-[2'-hydroxy-3'-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylquinoxalinyl-2-carboxamide;

[2'R-(2'R*, 3'R*,d'S*)]-N(8-butyl)-2-[2'-hydroxy-3'-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylquinolinyl-2-carboxamide;

The compounds of the present invention can be prepared according to theprocedures shown below in Reaction Scheme I. ##STR8## where:

q is 1 or 2; and

R, R², R¹ and X are as defined above.

Reaction Scheme I, above, is accomplished by carrying out the abovechemical reactions in sequential order. Once a reaction is complete, theintermediate compound may be isolated, if desired, by procedures knownin the art, for example, the compound may be crystallized and thencollected by filtration, or the reaction solvent may be removed byextraction, evaporation or decantation. The intermediate compound may befurther purified, if desired, by common techniques such ascrystallization or chromatography over solid supports such as silica gelor alumina, before carrying out the next step of the reaction scheme.

Reaction I.1 is a standard coupling reaction commonly employed in thesynthesis of peptides which is carried out by reacting a appropriatelysubstituted amine of formula IA, with an appropriately substitutedcarboxylic acid reactant of formula IB, in an aproticsolvent or mixtureof solvents. The reaction is carried out in the presence or absence of apromoting agent, preferably in the presence of a promoting agent, and inthe presence of a coupling reagent. Typical aprotic solvents for thisreaction are tetrahydrofuran and dimethylformamide, preferably a mixtureof such solvents. The reaction is carried out at a temperature fromabout --30° C. to about 25° C. The amine reactant is generally employedin equimolar proportions relative to the carboxylic acid reactant, inthe presence of an equimolar quantity to a slight excess of the couplingreagent. Typical coupling reagents include the carbodiimides such asdicyclohexylcarbodiimide (DCC) and N,N'-diethylcarbodiimide; theimidazoles such as carbonyldiimidazole; as well as reagents such asbis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP--C₁) orN-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ). A preferredcoupling reagent for this reaction is DCC. A promoting agent ispreferably included for this reaction; a preferred promoting agent ishydroxybenzotriazole hydrate (HOBT.H₂ O).

In reaction I.2, the compound prepared in reaction I.1 is oxidized usingusing standard procedures known in the art. For example, the compoundprepared in reaction I.1 is combined with an oxidizing agent in anorganic or aqueous solvent at a temperature from about -78° C. to about50° C. Typical oxidizing agents include hydrogen peroxide, sodiumiodate, potassium permanganate, osmium tetraoxide and peracids such asperacetic acid or meta-chloroperbenzoic acid. Typical solvents includewater or a halocarbon, such as methylene chloride or chloroform. Solventchoice is not critical so long as the solvent employed is inert to theongoing reaction and the reactants are sufficiently solubilized toeffect the desired reaction. The reaction is preferably carried out inmethylene chloride at a temperature from about -25° C. to about 25° C. Apreferred oxidizing agent is meta-chloroperbenzoic acid.

When preparing the compounds of formula I where R is hydrogen, it isnecessary to protect the amino group of reactant IB using a standardamino-protecting group. The amino-protecting group is removed after thecompound of formula I has been prepared. Preferred amino-protectinggroups are t-butoxycarbonyl (t-Boc) and benzyloxycarbonyl (Cbz).

The compounds of formula I where R is hydrogen may be acylated toprovide the compounds of formula I where R is formyl or C₂ -C₄ alkanoyl.The acylate may be accomplished with a suitable acyl halide, isocyanateor chloroformate, preferably in the presence of an acid scavenger suchas a tertiary amine, preferably triethylamine. The reaction is carriedout at a temperature of from about -20° C. to about 25° C. Typicalsolvents for this reaction include ethers and chlorinated hydrocarbons,preferably diethylether, chloroform or methylene chloride.

The compounds of the formula IA are useful, as described above, formaking the compounds of formula I. Compounds of formula IA where X is agroup having the formula ##STR9## can be prepared according to theprocedures shown below in Reaction Scheme A. ##STR10## where:

R^(b) is an amino-protecting group; and

R¹, R^(3a) and Y are as defined above.

Reaction Scheme A, above, is accomplished by carrying out the abovereactions 1-6 in sequential order. Once a reaction is complete, theintermediate compound may be isolated, if desired, by procedures knownin the art, for example, the compound may be crystallized and thencollected by filtration, or the reaction solvent may be removed byextraction, evaporation or decantation. The intermediate compound may befurther purified, if desired, by common techniques such ascrystallization or chromatography over solid supports such as silica gelor alumina, before carrying out the next step of the reaction scheme.

In Reaction A.1, the reaction is carried out by activating, that is,converting, a suitably substituted aryl, heterocycle or unsaturatedheterocycle carboxylic acid to the corresponding acyl chloride or acylbromide by reaction with thionyl chloride, thionyl bromide, phosphoroustrichloride, phosphorous tribromide, phosphorous pentabromide orphosphorous pentachloride according to procedures and under conditionsknown in the art. Suitable aryl, heterocycle or unsaturated heterocyclecarboxylic acid compounds are commercially available or prepared byprocedures known in the art.

In Reaction A.2, the acyl chloride or acyl bromide, prepared in ReactionA.1, is reacted with ammonia or a primary or secondary amine having thestructure: ##STR11## where R⁴, R⁵, R⁶ and p are as defined above forformula I, in a nonpolar aprotic solvent or mixture of solvents in thepresence or absence of an acid scavenger to afford the correspondingamide. The reaction is carried out at a temperature of from about -20°C. to about 25° C. Typical solvents for this reaction include ethers andchlorinated hydrocarbons, preferably diethylether, chloroform ormethylene chloride. Preferably, this reaction is carried out in thepresence of an acid scavenger such as a tertiary amine, preferablytriethylamine.

In Reaction A.3, the amide prepared in Reaction A.2, is reacted with astrong base in the presence of a solubilizing agent to afford thecorresponding anion which is then reacted in Reaction A.4 with a Weinrebamide to afford a ketone. Reaction A.3 is carried out in an aproticsolvent at a temperature of from about -78° C. to about 0° C. Typicalbases used in Reaction A.3 include lithium amide bases and alkyl lithiumbases, preferably C₁ -C₄ alkyllithium bases and lithium di(C₁-C₄)alkylamide bases. Typical solubilizing agents for Reaction A.3 aretetramethyl(C₁ -C₄)alkylenediamines, preferablytetramethylethylenediamine. Reaction A.4 is carried out in an aproticsolvent at a temperature from about -80° C. to about -40° C. Typicalsolvents for Reactions A.3 and A.4 include ethers, preferablytetrahydrofuran. In Reaction A.4, the anion is generally employed in anamount ranging from about equimolar proportions to about a three molarexcess of the anion, preferably in about a two molar excess of the anionrelative to the Weinreb amide reactant.

In Reaction A.5, the ketone prepared in Reaction A.3 is reduced to thecorresponding alcohol using a suitable reducing agent. The reaction iscarried out in a protic solvent at a temperature of from about -25° C.to about 25° C. Typical reducing agents for this reaction include sodiumborohydride, lithium borohydride, diisobutylaluminum hydride, and sodiumbis(2-methoxyethoxy)aluminum hydride. A preferred reducing agent issodium borohydride. Typical protic solvents for this reaction includealcohols, preferably ethanol.

Reaction A.6 is a standard amino deprotection reaction using proceduresand methods known in the art to afford the corresponding amine offormula IA. This amine is then reacted according to the proceduredetailed above in Reaction Scheme I. This amine may be reacted withoutpurification, but it is preferably purified first.

The compounds of formula IA where X is a group having the structure##STR12## are prepared according to the procedures shown below inReaction Scheme B. ##STR13## where R^(b), R¹, Y and R^(3b) are asdefined above.

Reaction Scheme B, above, is accomplished by carrying out reactions 1-7in sequential order. Once a reaction is complete, the intermediatecompound may be isolated by procedures known in the art, for example,the compound may be crystallized and then collected by filtration, orthe reaction solvent may be removed by extraction, evaporation ordecantation. The intermediate compound may be further purified, ifdesired, by common techniques such as crystallization or chromatographyover solid supports such as silica gel or alumina, before carrying outthe next step of the reaction scheme.

In Reaction B.1, a suitably substituted aryl or unsaturated heterocycleamine is protected, under standard conditions used with amino-protectinggroups known in the art. Reactions B.2 through B.5 are carried outsubstantially as described above in Reaction Scheme A.3-A.6, with theexception that in Reaction Scheme B, an additional deprotectionreaction, Reaction B.5, is necessary to remove the amino-protectinggroup introduced in Reaction B.1. This is a standard amino deprotectionreaction using procedures and methods known in the art. For example, thet-Boc group illustrated in Reaction Scheme II.1 may be removed using astrong acid, preferably trifluoroacetic acid.

In Reaction B.6, the illustrated intermediate is acylated with asuitable acyl halide, isocyanate or chloroformate, preferably in thepresence of an acid scavenger such as a tertiary amine, preferablytriethylamine. The reaction is carried out at a temperature of fromabout -20° C. to about 25° C. Typical solvents for this reaction includeethers and chlorinated hydrocarbons, preferably diethylether, chloroformor methylene chloride.

Reaction B.7 is a standard amino deprotection reaction using proceduresand methods known in the art to afford the corresponding amine IA, whichis used in Reaction Scheme I, above. This amine may be reacted withoutpurification, but it is preferably purified first.

The compounds of formula I where X is a group having the structure:##STR14## are prepared according to the procedures shown below inReaction Scheme C. ##STR15## where:

R¹, Y¹, R^(3a) and R^(b) are as defined above; and

G is halo.

Reaction Scheme C, above, is accomplished by carrying out reactions 1-9in sequential order. Once a reaction is complete, the intermediatecompound may be isolated, if desired, by procedures known in the art,for example, the compound may be crystallized and then collected byfiltration, or the reaction solvent may be removed by extraction,evaporation or decantation. The intermediate compound may be furtherpurified, if desired, by common techniques such as crystallization orchromatography over solid supports such as silica gel or alumina, beforecarrying out the next step of the reaction scheme.

In Reaction C.1, a β-lactone is formed by reacting amino-protectedserine with triphenylphosphine and diethylazodicarboxylate (DEAD) in anaprotic solvent at a temperature of from about -80° C. to 0° C. Thereaction is preferably carried out in an ether, such as tetrahydrofuranat a temperature of from about -800 C. to -50°C.

In Reaction C.2, the lactone ring prepared in reaction C.1 is opened byreacting the lactone with an appropriately substituted thioanion havingthe structure, --S--R¹, where R¹ is as defined above for formula I. Thethioanion compound is preferably formed by reacting the correspondingthiol with a strong base, such as sodium hydride or potassium hydride.The reaction is typically carried out in an aprotic solvent at atemperature from about 0° C. to about 40° C. and under an inertatmosphere, such as nitrogen. Typical solvents for this reaction includeethers, preferably tetrahydrofuran.

Reaction C.3 is carried out by activating, that is, converting, asuitably substituted compound prepared in Reaction C.2 to thecorresponding mixed arthydride under conditions known in the art. Forexample, the compound of formula from Reaction C.2 can be reacted withan C₁ -C₆ alkylchloroformate or benzylchloroformate, preferably in thepresence of an acid scavenger. Preferred acid scavengers are thetrialkylamines, preferably triethylamine. A preferred alkylchloroformatereactant is isobutylchloroformate. The reaction is typically carried outin an aprotic solvent such as ethyl acetate. Solvent choice is notcritical so long as the solvent employed is inert to the ongoingreaction and the reactants are sufficiently solubilized to effect thedesired reaction. The resulting mixed anhydride reactant is preferablyused in Reaction C.4 without further isolation or purification.

Reaction C.4 is accomplished in two steps. First, a solution of sodiumhydroxide, covered with a layer of an ether solvent, preferablydiethylether, is reacted with a large excess ofN-methyl-N-nitro-N-nitrosoguanidine to form a diazomethane reactant. Thesodium hydroxide is preferably used as an aqueous solution having aboutfour to six mol/liter of sodium hydroxide. Once this reaction issubstantially complete, the organic layer is dried over a dessicant suchas potassium hydroxide. This solution is then reacted with the mixedanthydride from Reaction C.3, above to form the corresponding α-diazocarbonyl compound. The diazomethane reactant is preferably used in thisreaction without isolation or purification. The reaction is typicallycarried out at a temperature of from about -50° C. to about -20° C.,preferably about -30° C.

In Reaction C.5, the α-diazo carbonyl compound prepared in Reaction C.4is reacted with an acid of the formula H--G where G is halo, in anaprotic solvent such as diethylether to form an α-halo carbonylcompound. A preferred acid reactant is hydrochloric acid which resultsin the α-chloro carbonyl compound. The reaction is typically carried outat a temperature from about -30° C. to about 0° C. Solvent choice is notcritical so long as the solvent employed is inert to the ongoingreaction and the reactants are sufficiently solubilized to effect thedesired reaction. The acid reactant is typically added in the form of ananhydrous gas in small increments until the reaction appearssubstantially complete. The reaction can be monitored by thin layerchromatography.

In Reaction C.6, the carbonyl moiety on the compound prepared inReaction C.5 is reduced using standard conditions known in the art toform the α-chloro hydroxy compound. For example, the compound preparedin Reaction C.5 is combined with a reducing agent in a mixture ofsolvents. Typical reducing agents include sodium borohydride, lithiumborohydride, zinc borohydride, diisobutylaluminum hydride or sodiumbis(2-methoxyethoxy)aluminum hydride. A preferred reducing agent issodium borohydride. Typical solvent mixtures include a protic andaprotic mixture such as tetrahydrofuran/water. Solvent choice is notcritical so long as the solvent employed is inert to the ongoingreaction and the reactants are sufficiently solubilized to effect thedesired reaction. The reaction is typically carried out at a temperaturefrom about -10° C. to about 10° C., preferably about 0° C.

In Reaction C.7, the α-chloro hydroxy compound prepared in Reaction C.6is treated with a strong base to form the corresponding epoxide understandard conditions known in the art. For example, the α-chloro hydroxycompound may be reacted with a potassium hydroxide/ethanol mixture in anorganic solvent such as ethyl acetate. Solvent choice is not critical solong as the solvent employed is inert to the ongoing reaction and thereactants are sufficiently solubilized to effect the desired reaction.The reaction is typically carried out at a temperature from about 0° C.to about the reflux temperature of the solvent. Preferably the reactionis carried out at room temperature.

In Reaction C.8, the epoxide prepared in Reaction C.7 is reacted with aheterocyclic reactant of the formula ##STR16## in a protic solvent at atemperature of from about 70° C. to 100° C. Solvent choice is notcritical so long as the solvent employed is inert to the ongoingreaction and the reactants are sufficiently solubilized to effect thedesired reaction. Typical solvents for this reaction include thealcohols, preferably ethanol. The reaction is preferably carried out ata temperature of about 80° C.

Reaction C.9 is a standard amino deprotection reaction using proceduresand methods known in the art to afford the corresponding amine, IA,which may be used in Reaction I, above.

The heterocyclic reactants, used in Reaction C.8 above, of the formula##STR17## can be prepared using procedures and methods known in the art.For example, the heterocyclic reactants were typically prepared from thecorresponding amino-protected amino acids by acid activation followed bytreatment with an alkylamine. This reaction is typically carried out inthe presence of an acid scavenger, such as N-methylmorpholine. Removalof the amino-protecting group using standard chemical deprotectingtechniques provides the heterocyclic reactants used above in ReactionC.8. Specifically, the[3S-(3R*,4aR*,8aR*)]-decahydroisoquinoline-3-N-t-butoxycarboxamide wasprepared using (2S)-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid bythe following procedure:

1) amino-protection (t-Boc);

2) acid activation/reaction with t-butylamine;

3) catalytic hydrogenation;

4) amino-deprotection.

The Weinreb amide used as a reactant in Reaction A.4 and B.3 can beprepared by reacting the compound prepared in Reaction scheme C.2 abovewith N-methoxy-N-methylamine in the presence of a promoting agent, anacid scavenger, and a coupling agent in an aprotic solvent or mixture ofsolvents at a temperature of from about -25° C. to 25° C. A preferredpromoting agent for this reaction is HOBT.H₂ O. Preferred acidscavengers are tertiary alkylamines, preferably triethylamine andN-methylmorpholine. A preferred coupling reagent is ethyldimethylaminopropylcarbodiimide hydrochloride. The Weinreb amideafforded by this reaction is preferably isolated prior to its use inReactions A.4 and B.3.

The carboxylic acid reactants used in the coupling reaction described inReaction I.1, to the extent not commercially available, are preparedusing procedures known in the art.

It will be understood by those skilled in the art that in performing theprocesses described above it may be desirable to introduce chemicalprotecting groups into the reactants in order to prevent secondaryreactions from taking place. Any amine, alkylamine or carboxy groupswhich may be present on the reactants may be protected using anystandard amino- or carboxy- protecting group which does not adverselyaffect the remainder of the molecule's ability to react in the mannerdesired. The various protective groups may then be removedsimultaneously or successively using methods known in the art.

As noted above, all asymmetric forms, individual isomers andcombinations thereof are considered part of this invention. Such isomersmay be prepared from their respective precursors by the proceduresdescribed above, or by resolving the racemic mixtures. The resolutioncan be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known in the art. Further details regardingresolutions can be found in Jacques et al., Enantiomers, Racemates, andResolutions, John Wiley & Sons 1981.

The compounds employed as initial starting material in the synthesis ofthe compounds of this invention are known and, to the extent notcommercially available are readily synthesized by standard procedurescommonly employed in the art.

The pharmaceutically acceptable salts of the invention are typicallyformed by reacting a compound of formula I with an equimolar or excessamount of acid or base. The reactants are generally combined in a mutualsolvent such as diethylether or benzene, for acid addition salts, orwater or alcohols for base addition salts. The salts normallyprecipitate out of solution within about one hour to about ten days andcan be isolated by filtration or other conventional methods.

The following Preparations and Examples further illustrate specificaspects of the present invention. It is to be understood, however, thatthese examples are included for illustrative purposes only and are notintended to limit the scope of the invention in any respect and shouldnot be so construed.

In the following Preparations and Examples, the terms melting point,nuclear magnetic resonance spectra, electron impact mass spectra, fielddesorption mass spectra, fast atom bombardment mass spectra, infraredspectra, ultraviolet spectra, elemental analysis, high performanceliquid chromatography, and thin layer chromatography are abbreviated"m.p.", "NMR", "EIMS", "MS (FD)", "MS (FAB)", "IR", "UV", "Analysis","HPLC", and "TLC", respectively. In addition, the absorption maximalisted for the IR spectra are only those of interest and not all of themaxima observed.

In conjunction with the NMR spectra, the following abbreviations areused: "s" is singlet, "d" is doublet, "dd" is doublet of doublets, "t"is triplet, "q" is quartet, "m" is multiplet, "dm" is a doublet ofmultiplets and "br.s", "br.d", "br.t", and "br.m" are broad singlet,doublet, triplet, and multiplet respectively. "J" indicates the couplingconstant in Hertz (Hz). Unless otherwise noted, NMR data refers to thefree base of the subject compound.

The NMR spectra were obtained on a Brudker Corp. 270 MHz instrument oron a General Electric QE-300 300 MHz instrument. The chemical shifts areexpressed in delta (δ) values (parts per million downfield fromtetramethylsilane). MS(FD) were taken on a Varian-MAT 731 Spectrometerusing carbon dendrite emitters. EIMS were obtained on a CEC 21-110instrument from Consolidated Electrodynamics Corporation. MS(FAB)spectra were obtained on a VG ZAB-3 spectrometer. IR spectra wereobtained on a Perkin-Elmer 281 instrument. UV spectra were obtained on aCary 118 instrument. TLC was carried out on E. Merck silica gel plates.Melting points are uncorrected.

Preparation 1 A. (2R)-2-N(t-Butoxycarbonyl)amino-3-naphth-2-ylthiopropanoic acid

To a solution of 2.14 g (13.4 mmol) 2-naphthalene thiol in 40 mL ofanhydrous tetrahydrofuran at room temperature, was added a suspension of0.54 g (13.5 mmol) of sodium hydride in mineral oil. After approximately15 minutes, a solution of 2.5 g (13.4 mmol) of(S)-N(t-butoxycarbonyl)-serine-β-lactone in 30 mL of tetrahydrofuran wasadded dropwise. The resultant reaction mixture was allowed to react forapproximately one hour and then was concentrated under reduced pressureto provide a gummy solid. This solid was purified using flashchromatagraphy (eluent of 1% methanol in ethyl acetate) to provide 4.35g of a white solid.

Yield: 94%.

¹ H NMR (CDCl₃): δ10.25 (s, 1H), 7.89 (s, 1H), 7.78 (m, 3H), 7.46 (m,3H), 5.39 (d, 1H), 4.61 (m, 1H), 3.49 (m, 2H), 1.37 (s, 9H).

B. (2R)-N(Methoxy)-N(methyl)[2-N(t-butoxycarbonyl)amino-3-naphth-2-ylthio]propanamide

To a cold (0° C.) solution containing 4.3 g (12.4 mmol) of the subtitledintermediate of Preparation 1A, 1.58 g (16.15 mmol)ofN,O-dimethylhydroxylamine hydrochloride, 2.18 g (16.15 mmol) of1-hydroxybenztriazole hydrate (HOBT.H₂ O), 2.24 mL (16.15 mmol) oftriethylamine and 2.73 mL (24.86 mmol) N-methylmorpholine in 100 mL ofmethylene chloride, was added 2.62 g (13.67 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Theresulting reaction mixture was allowed to react at room temperatureovernight. The reaction mixture was diluted with 100 mL of hexane,washed sequentially with 200 mL of a saturated sodium bicarbonatesolution and 200 mL of brine. The resulting layers were separated andthe organic layer was dried over sodium sulfate, filtered and thenconcentrated under reduced pressure to provide a clear yellow oil.

¹ H NMR (CDCl₃): δ7.90 (s, 1H), 7.80 (m, 3H), 7.49 (m, 3H), 5.41 (d,1H), 4.92 (m, 1H), 3.59 (s, 3H), 3.18-3.46 (m, 2H), 3.05 (s, 3H), 1.42(s, 9H).

MS (FD): m/e 391 (M⁺), 390 (100).

C. (3R)-N(t-Butyl)-2-[2'-oxo-3'-N(t-butoxycarbonyl)amino-4'-naphth-2-ylthio]butyl benzamide

To a cold (-78° C.) solution containing 8.60 g (45 mmol) ofN(t-butyl)-2-methylbenzamide and 14.2 mL (95 mmol) ofN,N,N',N'-tetramethylethylenediamine in 100 mL of anhydroustetrahydrofuran and under an inert atmosphere, was slowly added 111 mL(95 mmol) of a 0.85M solution of sec-butyllithium in hexanes, viasyringe. The internal temperature of the reaction vessel was monitoredduring the addition of the sec-butyllithium to ensure that thetemperature did not exceed -57° C. After allowing the resultant reactionmixture to react for approximately one hour at -78° C., a solution of7.90 g (20 mmol) of the subtitled intermediate of Example 1B in 80 mL oftetrahydrofuran was added dropwise. When the addition was complete, thereaction was warmed to -20° C. and then was quenched by the addition ofa saturated ammonium chloride solution. The resulting mixture was thendiluted with 600 mL of diethylether. The resulting layers were separatedand the organic layer was washed sequentially with a 1M sodium bisulfatesolution and a brine solution, dried over sodium sulfate, filtered andthen concentrated under reduced pressure to provide a yellow oil. Thisoil was purified using flash chromatography (gradient eluent of 10-50%ethyl acetate in hexane) to provide 8.5 g of the desired subtitledintermediate.

Yield: (82%).

¹ H NMR (CDCl₃): δ7.90 (s, 1H), 7.79 (t,3H), 7.48 (m, 3H), 7.40 (d, 1H),7.29 (m, 2H), 7.05 (d, 1H), 5.94 (br.s, 1H), 5.65 (m, 1H), 4.65 (d, 1H),4.24 (d, J=17 Hz, 1H), 3.86 (d, J=17 Hz, 1H), 3.66 (m, 1H), 3.40 (m,1H), 1.42 (s, 9H), 1.39 (s, 9H).

MS (FD): m/e 521 (M⁺), 521 (100).

D. [(2R-(2R*,3R*)]-N(t-Butyl)-2-[2'-hydroxy-3'-N(t-butoxycarbonyl)amino-4'-naphth-2-ylthio]butylbenzamide

To a solution of 3.49 g (6.7 mmol) of the subtitled intermediate ofPreparation 1C in 150 mL of absolute ethanol, was added 0.51 g (13 mmol)of sodium borohydride and the resulting reaction mixture was allowed toreact overnight at room temperature. The reaction was then cooled to 0°C., quenched with a saturated ammonium chloride solution and dilutedwith 550 mL of methylene chloride. The resulting layers were separatedand the organic layer was washed sequentially with 1N hydrochloric acid,2N sodium hydroxide and brine, dried over sodium sulfate, filtered andthen concentrated under reduced pressure to provide a colorless foam.This foam was purified using flash chromatography (gradient of 10-25%hexane in ethyl acetate) to provide 2.78 g of the desired subtitledintermediate.

Yield: 78%.

¹ H NMR (CDCl₃): δ7.84 (s, 1H), 7.73 (m, 3H), 7.41 (m, 3H), 7.29 (t,2H), 7.16 (t, 2H), 6.53 (s, 1H), 5.32 (d, 1H), 3.86 (m, 2H), 3.33 (m,2H), 2.83 (m, 2H), 1.40 (s, 9H).

MS (FD): m/e 523 (M⁺), 522 (100).

Analysis for C₃₀ H₃₈ N₂ O₄ S: Calcd: C, 68.94; H, 7.33; N, 5.36; Found:C, 68.65; H, 7.34; N, 5.15.

E. [(2R-(2R*, 3R*)]-N(t-Butyl)-2-[2'-hydroxy-3'-amino-4'-naphth-2-ylthio9 butyl benzamide

To a cold (0° C.) solution of 2.89 g (5.53 mmol) of the subtitledintermediate of Preparation 1D in 100 mL of methylene chloride, wasadded 18 mL of trifluoroacetic acid. The resulting reaction mixture wasallowed to react for approximately one hour. The reaction mixture wasthen concentrated under reduced pressure to provide a foam. This foamwas slurried in toluene and then concentrated under reduced pressure toprovide a foam which was purified using flash chromatography (eluent of5% methanol in methylene chloride) to provide 1.71 g of a white foam.

Yield: 74%.

¹ H NMR (CDCl₃): δ7.75-7.85 (m, 4H), 7.24-7.51 (m, 7H), 6.06 (s, 1H),3.75 (m, 1H), 3.61 (m, 1H), 3.07 (m, 2H), 2.95 (m, 2H), 1.47 (s, 9H) .

MS (FD): m/e 423 (M⁺), 422 (100).

Preparation 2 A. (2R)-2-N(t-Butoxycarbonyl)amino-3-phenylthio propanoicacid

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Preparation 1A.

Yield: 5.1 g (95%).

¹ H NMR (CDCl₃): δ7.43 (m, 2H), 7.22-7.34 (m, 3H), 5.31 (m, 1H), 4.54(m, 1H), 3.37-3.48 (m, 2H), 1.43 (s, 9H).

MS (FD): m/e 297 (M⁺), 297 (100).

B. (2R) -N(Methoxy)-N(methyl)[2-N(t-butoxycarbonyl)amino-3-phenylthio]propanamide

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Preparation 1B.

Yield: 4.40 g (79%).

¹ H NMR (CDCl₃): δ7.33 (m, 2H), 7.17 (m, 2H), 7.09 (m, 1H), 5.53 (d, J=9Hz, 1H), 4.73 (m, 1H), 3.45 (s, 3H), 3.19 (m, 1H), 2.95-3.06 (m, 4H),1.33 (s, 9H).

MS (FD): m/e 341 (M⁺), 340 (100).

C.(3R)-N(t-Butyl)-2-[2'-oxo-3'-N(t-butoxycarbonyl)amino-4'-phenylthio]butylbenzamide

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Preparation 1C.

Yield: 2.20 g (58%).

¹ H NMR (CDCl₃): 7.39 (m, 3H), 7.18-7.35 (m, 5H), 7.09 (d, J=6 Hz, 1H),6.00 (s, 1H), 5.63 (d, J=7 Hz, 1H), 4.56 (m, 1H), 4.20 (d, J=17 Hz, 1H),3.84 (d, J=17 Hz, 1H), 3.54 (m, 1H), 3.26 (m, 1H), 1.41 (s, 9H).

D.[(2R-(2R*,3R*)]-N(t-Butyl)-2-[2'-hydroxy-3'-N(t-butoxycarbonyl)amino-4'-phenylthio]butylbenzamide

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Preparation 1D.

Yield: 1.85 g (80%).

² H NMR (CDCl₃): δ7.17-7.45 (m, 9H), 6.04 (br.s, 1H), 5.08 (m, 1H), 3.87(m, 2H), 3.32 (m, 2H), 2.90 (m, 3H), 1.47 (d, 18H).

MS (FD): m/e 473 (M⁺), 472 (100).

Analysis for C₂₆ H₃₆ N₂ O₄ S: Calcd: C, 66.07; H, 7.68; N, 5.93; Found:C, 66.09; H, 7.75; N, 5.86.

E. [(2R-(2R*,3R*)]-N(t-Butyl)-2-[2'-hydroxy-3'-amino-4'-phenylthio]butylbenzamide

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Preparation 1E.

Yield: 1.00 g (81%).

¹ H NMR (CDCl₃): δ7.19-7.43 (m, 9H), 6.04 (br.s, 1H), 5.43 (br.s, 1H),3.71 (m, 1H), 3.47 (m, 1H), 2.83 -3.01 (m, 4H), 1.47 (s, 9H) .

MS (FD): m/e 373 (M⁺), 372, 373 (100).

Preparation 3 A. (2R)-N(Benzyloxycarbonyl)amino-3-naphth-2-ylthiopropanoic acid

To a solution of 1.28 g (8.00 mmol) of naphthalene-2-thiol in 30 mLtetrahydrofuran, was slowly added 1.77 g (8.16 g) of 60% sodium hydride,under nitrogen. After stirring for approximately 15 minutes,N(benzyloxycarbonyl)serine-β-lactone in 20 mL of tetrahydrofuran wasslowly added. The reaction mixture was allowed to react at roomtemperature for approximately one hour, and then was concentrated underreduced pressure to provide a residue. This residue was dissolved inethyl acetate and washed sequentially with 0.5N sodium bisulfate and asaturated brine solution. The resulting layers were separated and theorganic layer was dried over sodium sulfate, filtered, and thenconcentrated under reduced pressure to provide a residue. This residuewas purified using flash chromatography to provide 2.08 g of a paleyellow solid.

Yield: 68%.

¹ H NMR (CDCl₃): δ3.42-3.61 (br.m, 2H), 5.53-5.76 (br.s, 1H), 4.85-5.08(br.m, 2H) , 5.54-5.76 (hr.s, 1H), 7.06-7.97 (m, 12H) .

[α]_(D) -55.72° (C 1.0, MeOH).

IR (KBr): 3348, 3048, 1746, 1715, 1674, 1560, 1550, 1269, 1200, 1060cm⁻¹.

MS (FD): m/e 381 (M⁺), 381 (100).

Analysis for C₂₀ H₁₉ NO₄ S: Calcd: C, 66.12; H, 5.02; N, 3.67; Found: C,66.22; H, 5.04; N, 3.86.

B. (3R) Benzyl 2-aza-3-(naphth-2-ylthiomethyl)-4-oxo-5-diazo pentanoate

To a cold (-30° C.) solution of 15.38 g (40.3 mmol) of the subtitledintermediate from Preparation 3A in 230 mL of ethyl acetate and undernitrogen, was slowly added 5.62 mL (40.3 mmol) of triethylamine, viasyringe. To the resulting solution was then added 7.84 mL (60.5 mmol) ofisobutyl chloroformate, via syringe. In a separate flask, 10 g ofN(methyl)-N(nitro)-N(nitroso)guanidine was carefully added to a bilayermixture of 170 mL of diethylether and 170 mL of a 5N sodium hydroxidesolution, resulting in a large evolution of gas. When this reaction wassubstantially complete, the organic layer was decanted from the aqueouslayer onto potassium hydroxide and dried. This diazomethane formationand addition was repeated using identical quantities of diethylether andsodium hydroxide and 30 g of N(methyl)-N(nitro)-N(nitroso)guanidine. Theresultant diazomethane reactant was then added to the mixed anhydridesolution prepared above and the reaction mixture was allowed to reactcold (-30° C.) for approximately 20 minutes. When the reaction wassubstantially complete, as indicated by TLC, nitrogen was bubbledthrough the solution using a fire polished Pasteur pipet to remove anyexcess diazomethane and then the solution was concentrated under reducedpressure to provide a residue. This residue was purified using flashchromatography (eluent of 10% ethyl acetate in methylene chloride) toprovide 13.62 g of a yellow oil.

Yield: 83%.

¹ H NMR (CDCl₃): δ3.32-3.46 (m, 2H), 4.40-4.67 (m, 1H), 5.00-5.09 (m,2H), 5.44 (s, 1H), 5.76 (d, J=7.8 Hz, 1H), 7.25-7.86 (m, 12H) .

C. (3.R) Benzyl 2-aza-3-(naphth-2-ylthiomethy)-4-oxo-5-chloro pentanoate

A short burst (about 2 seconds) of anhydrous hydrochloric acid (gas) waspassed through a cold (-20° C.) solution of 13.62 g (33.59 mmol) of thesubtitled intermediate from Preparation 3B in 230 mL of diethylether,resulting in the evolution of a gas. This procedure was repeated takingcare not to add excess hydrochloric acid. When the reaction wassubstantially complete, as indicated by TLC, the solution wasconcentrated under reduced pressure to provide a residue. This residuewas purified using flash chromatography (eluent of 10% ethyl acetate inmethylene chloride) to provide 12.05 g of a pale tan solid.

Yield: 87%.

¹ H NMR (CDCl₃): δ3.41 (dd, J=12,6 Hz, 1H), 3.53 dd, J=12,6 Hz, 1H),4.18 AB q, Δυ=41.9 Hz, J=15.9 Hz, 2H), 4.77 dd, J=9, 3 Hz, 1H), 5.04 ABq, J=12 Hz, Δυ=10.4 Hz, 2H), 5.59 d, J=7 Hz, 1H), 7.24-7.85 (complex,12H) .

[α]_(D) -80.00° (C 1.0, MeOH).

IR (CHCl₃): 3426, 3031, 3012, 1717, 1502, 1340, 1230, 1228, 1045 cm⁻¹.

MS (FD): m/e 413 (M⁺), 413 (100).

Analysis for C₂₂ H₂₀ NO₃ SCl: Calcd: C, 63.84; H, 4.87; N, 3.38; Found:C, 64.12; H, 4.95; N, 3.54.

D. 3R-(3R*,4S*)] Benzyl2-aza-3-(naphth-2ylthiomethyl)-4-hydroxy-5-chloro pentanoate

To a cold (0° C.) solution of 530 mg (1.28 mmol) of the subtitledintermediate of Preparation 3C, in 10 mL of tetrahydrofuran and 1 mL ofwater, was added 73 mg (1.92 mmol) of sodium borohydride. When thereaction was substantially complete as indicated by TLC, the solutionwas adjusted to pH 3 using 10 mL of an aqueous saturated ammoniumchloride solution and 500 μL of a 5N hydrochloric acid solution. Theresultant solution was extracted twice with methylene chloride and thecombined organic layers were washed with water, dried over sodiumsulfate, filtered and then concentrated under reduced pressure toprovide a residue. This residue was purified using radial chromatography(eluent of methylene chloride) to provide 212 mg of a tan solid.

Yield: 40%.

¹ H NMR (CDCl₃): δ3.40 (s, 2H), 3.61-3.71 (m, 2H), 3.97-3.99 (m, 2H),4.99 (s, 2H), 5.16 (br.s, 1H), 7.21-7.83 (complex, 12H).

MS (FD): m/e 415 (M⁺), 415 (100).

[α]_(D) -47.67° (C 0.86, MeOH).

IR (CHCl₃): 3630, 3412, 3011, 1720, 1502, 1236, 1044 cm⁻¹.

Analysis for C₂₂ H₂₂ NO₃ ClS: Calcd: C, 63.53; H, 5.33; N, 3.37; Found:C, 63.72; H, 5.60; N, 3.64.

E. [3R-(3R*,4S*) ] Benzyl 2-aza-3-oxiranyl-4-naphth-2-ylthio butanoate

A solution of 31 mg (0.55 mmol) of potassium hydroxide in 1 mL ofethanol was added to a solution of 190 mg (0.46 mmol) of the subtitledintermediate of Preparation 3D, in 6 mL of a 1:2 ethanol/ethyl acetatesolution. When the reaction was substantially complete, as indicated byTLC, the reaction mixture was poured into a water/methylene chloridemixture. The resulting layers were separated, and the organic layer waswashed with water, dried over sodium sulfate, filtered and thenconcentrated under reduced pressure to provide a residue. This residuewas purified using radial chromatography (eluent of 10% ethyl acetate inmethylene chloride) to provide 172 mg of a light tan solid.

Yield: 99%.

¹ H NMR (CDCl₃): δ2.76 (br.s, 2H) 3.01 (br.s, 1H), 3.31 (d, J=5 Hz, 2H),3.77 (br.s, 1H), 5.05 (s, 2H), 5.22 (d, J=6 Hz, 1H), 7.25-7.85 (complex,12H) .

[a]_(D) -125.42° (C 0.59, MeOH).

MS (FD): m/e 379 (M⁺), 379 (100).

IR (CHCl₃): 3640, 3022, 2976, 1720, 1502, 1235, 1045 cm⁻¹.

Analysis for C₂₂ H₂₁ NO₃ S: Calcd: C, 69.63; H, 5.58; N, 3.69; Found: C,69.41; H, 5.53; N, 3.64.

F. [3R-(3R*,4R*,3'S*,4a'S*,8a'S*)] Benzyl,[2-aza-3-)naphth-2-ylthiomethyl)-4-hydroxy-5-(3'-(1"-N(t-butyl)amino-1"-oxomethyl)octahydroisoquinolin-2'-yl)]pentanoate

A solution was prepared containing 165 mg (0.40 mmol) of the subtitledintermediate of Preparation 3E and 94 mg (0.43 mmol) of3-(1-N(t-butyl)amino-1-oxomethyl)octahydro-(2H)-isoquinoline in 5 mL ofethanol. The resulting reaction mixture was allowed to react at 80° C.for approximately 19 hours. The solution was then cooled to roomtemperature and concentrated under reduced pressure to provide aresidue. This residue was purified using radial chromatography (eluentof 10% ethyl acetate in methylene chloride) to provide 103 mg of anoff-white foam.

Yield: 42%.

¹ H NMR (CDCl₃): δ1.10-1.73 (m, 20H), 2.13-2.31 (m, 2H), 2.44-2.53 (m,1H), 2.56-2.68 (m, 1H), 2.86-2.97 (m, 1H), 3.52 (br.s, 2H), 4.02 (br.s,2H), 4.98 (s, 2H), 5.65 (s, 1H), 5.94 (s, 1H), 7.25-7.83 (complex, 13H).

MS (FD): m/e 629 (M⁺), 138 (100).

[α]_(D) -92.45° (C 1.06, MeOH).

IR (CHCl₃): 3429, 3010, 2929, 1713, 1670, 1514, 1455, 1047 cm⁻¹.

Analysis for C₃₅ H₄₇ N₃ O4S: Calcd: C, 69.98; H, 7.67; N, 6.80; Found:C, 69.86; H, 7.78; N, 6.58.

G.[2R-(2R*,3R*,3'S*,4a'S*,8a'S*)]-N(t-butyl)-2'-[2-hydroxy-3-amino-4-(naphth-2-ylthio)]butyloctahydroisoquinoline-3'-carboxamide

A solution was prepared containing 50 mg (0.081 mmol) of the subtitledintermediate of Preparation 3F and 1 mL of a 38% aqueous hydrobromicacid solution in acetic acid. The resultant reaction mixture was allowedto react at room temperature for approximately 1 hour and then wasconcentrated under reduced pressure to provide a residue. This residuewas slurried with toluene and then concentrated under reduced pressureto provide 61 mg of the desired subtitled intermediate. This compoundwas used crude without purification in Example 9.

¹ H NMR (CDCl₃): δ1.14 (s, 1H), 1.17-2.07 (complex, 15H), 2.66-2.87 (m,2H), 3.21-3.25 (m, 2H), 3.75 (d, J=12 Hz, 1H), 3.85 (d, J=6 Hz, 1H),4.36-4.47 (m, 1H), 6 .73 (s, 1H), 7.39-7.90 (complex, 7H).

MS (FD): 483 (M⁺), 483 (100).

Preparation 4 A. (2R)-2-N(benzyloxycarbonyl)amino-3 -phenylthiopropanoic acid

The desired subtitled intermediate was prepared substantially inaccordance with the procedure detailed in Procedure 3A, using 13.1 mL(127 mmol) of thiophenol, 4.6 g (117 mmol) of a 60% sodium hydridesolution and 25.6 g (116 mmol) of (L)-N(benzyloxycarbonyl)serineβ-lactone in 450 mL of tetrahydrofuran to provide a residue. Thisresidue was purified using flash chromatography (gradient eluent of 0-2%acetic acid in a 4:1 methylene chloride/ethyl acetate mixture to provide27.9 g of a white solid.

Yield: 72%.

¹ H NMR CDCl₃): δ7.55-7.18 (m, 10H), 5.55 (d, J=7 Hz, 1H), 5.08 (s, 2H),4.73-4.60 (m, 1H), 3.55-3.30 (m, 2H).

IR (KBr): 3304, 3035, 1687, 1532, 736 cm¹.

MS (FD): m/e 332, 288, 271, 181.

Analysis for C₁₇ H₁₇ NO₄ S: Calcd: C, 61.61; H, 5.17; N, 4.23; Found: C,61.69; H, 5.22; N, 4.47.

B. (3R) Benzyl 2-aza-3-(phenylthiomethyl)-4-oxo-5-diazo pentanoate

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Procedure 3B, using 12.1 g (37 mmol) ofthe subtitled compound of Preparation 4A, 5.09 mL (37 mmol) oftriethylamine, 7.13 mL (55 mmol) of isobutyl chloroformate, 146 mmol ofa diazomethane solution to provide a residue. The diazomethane solutionwas prepared using 100 mL of diethylether, 150 mL of a 5N sodiumhydroxide solution and 21 g (146 mmol) ofN(methyl)-N(nitro)-N(nitroso)-guanidine as described in Preparation 3B.This residue was purified using flash chromatography (gradient eluent of0-5% ethyl acetate in methylene chloride) to provide a yellow oil.

Yield: 73%.

¹ H NMR (CDCl₃): δ7.50-7.19 (m, 10H), 5.62 (d, J=7 Hz, 1H), 5.47 (br.s,1H), 5.11 (s, 2H), 4.50-4.32 (m, 1H), 3.33 (d, J=6 Hz, 1H).

IR (KBr): 3012, 2115, 1720, 1501, 1367, 1228 cm⁻¹.

MS (FD): m/e 356, 328, 242.

C. (3R) Benzyl, 2-aza-3-(phenylthiomethyl)-4-oxo-5-chloro pentanoate

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Procedure 3C, using 22.3 g (63 mmol) ofthe subtitled compound of Preparation 4B and small quantities ofhydrochloric acid (gas) in 400 mL of diethylether to provide 21 g of awhite solid. This solid was used without further purification.

¹ H NMR (CDCl₃): δ7.50-7.15 (m, 10H), 5.56 (dd, J=2,6.7 Hz, 1H), 5.11(s, 2H), 4.78-4.67 (m, 1H), 4.20 (d, J=15.9 Hz, 1H), 4.12 (d, J=15.9 Hz,1H), 3.48-3.23 (m, 2H) .

IR (KBr): 3349, 1732, 1684, 1515, 1266 cm⁻¹.

MS (FD): m/e 363 (M⁺).

Analysis for C₁₈ H₁₈ NO₃ SCl: Calcd: C, 59.42; H, 4.99; N, 3.85; Found:C, 59.57; H, 5.09; N, 4.13.

D. [3R-(3R*, 4S*) ] Benzyl 2-aza-3-(phenylthiomethyl)-4-hydroxy-5-chloropentanoate

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Procedure 3D, using 21 g (58 mmol) of thesubtitled compound of Preparation 4C, 2.4 g (63 mmol) of sodiumborohydride in 300 mL of tetrahydrofuran to provide a residue. Thisresidue was purified using flash chromatography (gradient eluent of 0-2%methanol in methylene chloride) followed by flash chromatography(gradient eluent of 0-2% ethyl acetate in chloroform) and thenrecrystallized from methylene chloride at -78° C. to provide 8.3 g ofthe subtitled compound.

Yield: 39%.

¹ H NMR (CDCl₃): δ7.47-7.19 (m, 10H), 5.22-5.03 (m, 1H), 5.09 (s, 2H),4.01-3.89 (m, 2H), 3.75-3.58 (m, 2H), 3.32 (d, J=4 Hz, 2H).

IR (KBr): 3321, 2951, 1688, 1542, 1246, 738 cm⁻¹.

MS (FD): m/e 366 (M⁺), 119.

Analysis for C₁₈ H₂₀ NO₃ SCl: Calcd: C, 59.09; H, 5.51; N, 3.83; Found:C, 59.03; H, 5.50; N, 3.96.

E. [3R-(3R*,4S*)] Benzyl 2-aza-3-oxiranyl-4-phenylthio butanoate

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Procedure 3E, using 8.3 g (23 mmol) ofthe subtitled compound of Preparation 4D, 1.4 g (25 mmol) of potassiumhydroxide in 400 mL of ethanol to provide a residue. This residue waspurified using flash chromatography (gradient eluent of 0-2% ethylacetate in methylene chloride) to provide 6.4 g of a white solid.

Yield: 85%.

¹ H NMR (CDCl₃): δ7.45-7.15 (m, 10 H), 5.12 (s, 1H), 5.08 (s, 2H),3.77-3.62 (m, 1H), 3.21 (d, J=6 Hz, 2H), 2.99 (m, 1H), 2.77 (m, 2H) .

IR (KBr): 3303 ,3067, 1694, 1538, 1257, 741 cm⁻¹.

MS (FD) m/e 329.

Analysis for C₃₂ H₄₅ N₃ O₄ S: Calcd: C, 65.63; H , 5.81; N, 4.25; Found:C, 65.48; H, 5.82; N, 4.29.

F. [3R-(3R*,4R*,3'S*,4a'S*,8a'S*)] Benzyl,[2-aza-3-phenylthiomethy)-4-hydroxy-5-(3'-(1"-N(t-butyl)amino-1"-oxomethyl)octahydroisoquinolin-2'-yl)]pentanoate

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Procedure 3F, using 6.3 g (19 mmol) ofthe subtitled compound of Preparation 4E, 5 g (21 mmol) of3-[N(t-butyl)aminocarbonyl]octohydro-(2H)-isoquinoline in 300 mL ofethanol to provide a residue. This residue was purified using flashchromatography (gradient eluent of 0-20% ethyl acetate in methylenechloride) to provide 4.3 g of a white solid.

Yield: 40%.

¹ H NMR (CDCl₃): δ7.41-7.11 (m, 10H), 5.90 (d, J=5 Hz, 1H), 5.64 (s,1H), 5.05 (d, J=4 Hz, 2H), 4.08-3.90 (m, 2H), 3.40 (d, J=6, 2H), 3.05(s, 1H), 2.95-2.85 (m, 1H), 2.62-2.45 (m, 2H), 2.28-2.15 (m, 2H),2.05-1.88 (m, 2H), 1.78-1.10 (m, 7H), 1.29 (s, 9H) .

IR(KBr): 3330, 2925, 2862, 1706, 1661, 1520, 1454, 1246, 738, 694 cm⁻¹.

MS (FD): m/e 568 (M⁺), 467.

Analysis for C₃₂ H₄₅ N₃ O₄ S: Calcd: C, 67.69; H, 7.99; N, 7.40; Found:C, 67.64; H, Q 20; N, 7.45.

G.[2R-(2R*,3R*,3'5*,4a'5*,8a'5*)]-N(t-butyl)-2'-[2-hydroxy-3-amino-4-(phenylthio)]butyloctohydroisoquinoline-3'-carboxamide

The desired subtitled compound was prepared substantially in accordancewith the procedure detailed in Procedure 3G using 1 g (1.8 mmol) of thesubtitled compound of Preparation 4F and 40 mL of a 30% hydrobromic acidin acetic acid solution, with the exception that the crude material wasdissolved in 30 mL of methanol. To the resulting solution, was added 2mL of diethylamine and 2 mL of concentrated ammonium hydroxide and thenthe mixture was concentrated under reduced pressure to provide aresidue. This residue was redissolved in water and ethyl acetate. Theresulting layers were separated and the organic layer was washedsequentially with an aqueous sodium bicarbonate solution and brine,dried over sodium sulfate, filtered and then reduced to dryness underreduced pressure to provide a residue. This residue was purified usingflash chromatography (gradient eluent of 0-10% methanol in chloroform(containing 3 drops of ammonium hydroxide per 1000 mL of chloroform)) toprovide 0.54 g of a white foam.

Yield: 71%.

¹ H NMR (CDCl₃): δ7.41-7.16 (m, 5H), 6.07 (s, 1H), 3.78-3.70 (m, 1H),3.45-3.38 (m, 1H), 3.03-2.84 (m, 3H), 2.38-2.20 (m, 3H), 2.00-1.05 (m,12H), 1.33 (s, 9H).

IR (KBr): 2924, 2862, 1660, 1517, 1454, 1439, 737, 691 cm⁻¹.

MS (FD): m/e 434 (M⁺), 293.

EXAMPLE 1 A.[2'R-(2'R*,3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3'-naphth-2-ylthiomethyl-4'-aza-5',8'-dioxo-6'-(N(t-butoxycarbonyl)amino-8'-amino]octylbenzamide

To a solution of 0.50 g (1.18 mmol) of the subtitled intermediate ofPreparation 1E in 11.5 mL of a 10:1.5 solution oftetrahydrofuran/dimethylformamide, was added 71.5 mg 0.289 g (1.24 mmol)of (2S)-2-N(t-butoxycarbonyl-3-carbamoyl propanoic acid, and 0.163 g(1.21 mmol) of HOBT.H₂ O. This mixture was cooled to -10° C. beforeadding and 0.249 g (1.21 mmol) of dicyclohexylcarbodiimide (DCC). Afterapproximately one hour, the reaction mixture was warmed to roomtemperature and allowed to react overnight. The reaction was then cooledto 0° C. and filtered to remove a precipitate. The filtrate wasconcentrated under reduced pressure to provide a residue which wasredissolved in ethyl acetate and washed sequentially with a saturatedsodium bicarbonate solution, water, a 5% citric acid solution and brine.The resulting layers were separated and the organic layer was dried oversodium sulfate, filtered and then concentrated under reduced pressure toprovide white solid. This solid was purified using flash chromatography(gradient eluent of 1-5% methanol in methylene chloride) to provide 0.64g of the desired subtitled compound.

Yield: 85%.

¹ H NMR (CDCl₃): δ7.71-7.81 (m, 4H), 7.17-7.48 (m, 7H), 6.38 (br.s, 1H),6.25 (br.s, 1H), 5.98 (m, 1H), 5.81 (br.s, 1H), 4.41 (m, 1H), 4.19 (m,1H), 3.87 (m, 1H), 3.44 (m, 1H), 3.27 (m, 1H), 2.52-2 .91 (m, 4H), 1.43(s, 9H).

MS (FD): m/e 637 (M⁺), 636 (100).

Analysis for C₃ H₄₄ N₄ O₆ S: Calcd: C, 64.13; H, 6.96; N, 8.80; Found:C, 64.09; H, 6.90; N, 8.81.

B.[2'R-(2'R*,3'S*)]-N(t-butyl)-2-[2'-hydroxy-3'-naphth-2-ylthiomethyl-4'-aza-5',8'-dioxo-6',8'-diamino]octylbenzamide

The subtitled compound was prepared substantially in accordance with theprocedure detailed in Preparation 1E using 0.64 g (1.00 mmol) of thesubtitled intermediate of Example 1A in 10 mL of 15% trifluoroaceticacid in methylene chloride to provide a white foam. This foam waspurified using flash chromatography (gradient eluent of 5-10% methanolin methylene chloride) to provide a white solid.

¹ H NMR (CDCl₃): δ7.71-7.93 (m, 4H), 7.17-7.47 (m, 7H), 6.18-6.39 (m,2H), 5.71 (m, 1H), 4.23 (m, 1H), 3.89 (m, 1H), 3.59 (m, 1H), 3.33-3.45(m, 3H), 2.76-2.97 (m, 2H), 2.60 (m, 1H), 1.80-2.26 (m, 4H), 1.45 (s,9H).

MS (FD): m/e 537 (M⁺), 536 (100 ) .

C. [2'R-(2'R*,3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3'-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylbenzamide

To a solution of 0.10 g (0.19 mmol) of the subtitled intermediate ofExample 1B, in 10 mL of methylene chloride, was added 0.019 g (0.19mmol) acetic anhydride dropwise. The resulting reaction mixture wasallowed to react for approximately four hours and then was diluted withmethylene chloride, and washed sequentially with 1N aqueous hydrochloricacid and brine. The resulting layers were separated and the organiclayer was dried over sodium sulfate, filtered and then concentratedunder reduced pressure to provide a clear oil which solidified onstanding. This oil was purified using flash chromatography (eluent of 5%methanol in methylene chloride) to provide 0.069 g of a white foam.

Yield: 57%.

¹ H NMR (CDCl₃): δ7.71-7.81 (m, 4H), 7.19-7.48 (m, 8H), 7.01 (d, J=7 Hz,1H), 5.95 (m, 3H), 5.40 (m, 1H), 4.62 (m, 1H), 4.23 (m, 1H), 3.86 (m,1H), 3.28-3.47 (m, 2H), 2.78-2.98 (m, 3H), 2.49 (m, 1H), 1.85 (s, 3H),1.48 (s, 9H).

MS (FD): m/e 579 (M⁺), 578 (100).

Analysis for C₃₁ H₃₈ N₄ O₅ S: Calcd: C, 64.34; H, 6.62; N, 9.68; Found:C, 64.33; H, 6.66; N, 9.44.

EXAMPLE 2[2'R-(2'R*,3'R*,6'S*)]-N(t-Butyl)-2-[2'-hydroxy-3'-phenylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylbenzamide

The desired titled compound was prepared substantially in accordancewith the procedure detailed in Example 1, using the subtitledintermediate of Preparation 2E and (2S)-2-N(t-butoxycarbonyl-3-carbamoylpropanoic acid.

Yield: 95 mg (98%).

¹ H NMR (CDCl₃): δ7.06-7.43 (m, 11H), 5.89-6.06 (m, 3H) , 5.45 (m, 1H),4.66 (m, 1H), 4.19 (m, 1H), 3.86 (m, 1H), 3.22 -3.37 (m, 2H), 2.80-2.98(m, 3H), 2.54 (m, 1H), 2.02 (m, 3H), 1.47 (s, 9H).

MS (FD): m/e 529 (M⁺), 528 (100).

Analysis for C₂₇ H₃₆ N₄ O₅ S: Calcd: C, 61.34; H, 6.86; N, 10.60; Found:C, 61.21; H, 6.96; N, 10.33.

As noted above, the compounds of the present invention are useful forinhibiting HIV protease which is associated with vital componentproduction and assembly. An embodiment of the present invention is amethod of treating or preventing HIV infection comprising administeringto a primate in need thereof an effective amount of a compound offormula I or a pharmaceutically acceptable salt thereof. Anotherembodiment of the present invention is a method of treating orpreventing AIDS comprising administering to a primate in need thereof aneffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof. A further embodiment of the present inventionis a method of inhibiting HIV replication comprising administering to anHIV infected cell, a cell susceptible to HIV infection or a primate inneed thereof, an effective amount of a compound of formula I or apharmaceutically acceptable salt thereof.

The term "effective amount" as used herein, means an amount of acompound of the present invention which is capable of inhibiting the HIVprotease mediated viral component production and assembly. The HIVprotease inhibition contemplated by the present method includes eithertherapeutic or prophylactic treatment, as appropriate. The specific doseof compound administered according to this invention to obtaintherapeutic and/or prophylactic effects will, of course, be determinedby the particular circumstances surrounding the case, including, forexample, the compound administered, the route of administration, thecondition being treated and the individual being treated. A typicaldaily dose will contain a dosage level of from about 0.01 mg/kg to about50 mg/kg of body weight of an active compound of this invention.Preferred daily doses generally will be from about 0.05 mg/kg to about20 mg/kg and ideally from about 0.1 mg/kg to about 10 mg/kg.

The compounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular andintranasal. The compounds of the present invention are preferablyformulated prior to administration. Therefore, another embodiment of thepresent invention is a pharmaceutical formulation comprising aneffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier,diluent or excipient therefor.

The active ingredient in such formulations comprises from 0.1% to 99.9%by weight of the formulation. By "pharmaceutically acceptable" it ismeant that the carrier, diluent or excipient is compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

The present pharmaceutical formulations are prepared by known proceduresusing known and readily available ingredients. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), ointments containing, for example, up to10% by weight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powdersand the like.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. The term"active ingredient" means a compound according to formula I or apharmaceutically acceptable salt thereof.

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

    ______________________________________                                                       Quantity                                                                      (mg/capsule)                                                   ______________________________________                                        Active ingredient    250                                                      Starch, dried        200                                                      Magnesium stearate   10                                                       Total                460     mg                                               ______________________________________                                    

Formulation 2

A tablet is prepared using the ingredients below:

    ______________________________________                                                         Quantity                                                                      (mg/capsule)                                                 ______________________________________                                        Active ingredient     250                                                     Cellulose, microcrystalline                                                                         400                                                     Silicon dioxide, fumed                                                                              10                                                      Stearic acid          5                                                       Total                 665     mg                                              ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3

An aerosol solution is prepared containing the following components:

    ______________________________________                                                         Weight                                                       ______________________________________                                        Active ingredient  0.25                                                       Methanol           25.75                                                      Propellant 22      70.00                                                      (Chlorodifluoromethane)                                                       Total              100.00                                                     ______________________________________                                    

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to -30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets, each containing 60 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient    60 mg                                                    Starch               45 mg                                                    Microcrystalline cellulose                                                                         35 mg                                                    Polyvinylpyrrolidone 4 mg                                                     (as 10% solution in water)                                                    Sodium carboxymethyl starch                                                                        4.5 mg                                                   Magnesium stearate   0.5 mg                                                   Talc                 1 mg                                                     Total                150 mg                                                   ______________________________________                                    

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The aqueous solution containingpolyvinylpyrrolidone is mixed with the resultant powder, and the mixturethen is passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50° C and passed through a No. 18 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc, previouslypassed through a No. 60 mesh U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yield tabletseach weighing 150 mg.

Formulation 5

Capsules, each containing 80 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient   80 mg                                                     Starch              59 mg                                                     Microcrystalline cellulose                                                                        59 mg                                                     Magnesium stearate  2 mg                                                      Total               200 mg                                                    ______________________________________                                    

The active ingredient, cellulose, starch and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

Formulation 6

Suppositories, each containing 225 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient    225 mg                                                   Saturated fatty acid glycerides                                                                    2,000 mg                                                 Total                2,225 mg                                                 ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions, each containing 50 mg of active ingredient per 5 mL dose,are made as follows:

    ______________________________________                                        Active ingredient        50 mg                                                Sodium carboxymethyl cellulose                                                                         50 mg                                                Syrup                     1.25 mL                                             Benzoic acid solution     0.10 mL                                             Flavor                   q.v.                                                 Color                    q.v.                                                 Purified water to total   5 mL                                                ______________________________________                                    

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with aportion of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

Formulation 8

An intravenous formulation may be prepared as follows:

    ______________________________________                                        Active ingredient     100    mg                                               Isotonic saline       1,000  mL                                               ______________________________________                                    

The solution of the above ingredients generally is administeredintravenously to a subject at a rate of 1 mL per minute.

The following experiment (Fluorescence HIV-1 Protease Inhibitor Assay)was carried out to demonstrate the ability of the compounds of thepresent invention to inhibit HIV protease.

As used herein, the abbreviations are defined as follows:

BSA--bovine serum albumin

BOC--t-butyloxycarbonyl

BrZ--2-bromobenzyloxycarbonyl

2-ClZ--- 2-chlorobenzyloxycarbonyl

DCC--dicyclohexylcarbodiimide

DIEA--diisopropylethylamine

DTT--dithiothreitol

EDTA--ethylenediaminetetraacetic acid

PITC--fluorescein isothiocarbamyl

HEPES--4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid

MES--4 morpholineethanesulfonic acid

PAM--phenylacetimidomethyl

TAPS--3-[tris(hydroxymethyl)methyl]amino-1-sulfonic acid

TRIS--tris(hydroxymethyl)aminomethane

TOS--p-toluenesulfonyl (tosyl)

I. Preparation of Protease and Gag Fractions A. Culture of E. coli K12L507/pHP10D

Lyophils of E. coli K12 L507/pHP10D were obtained from the NorthernRegional Research Laboratory, Peoria, Ill. 61604, under the accessionnumber NRRL B-18560 (deposited Nov. 14, 1989). The lyophils weredecanted into tubes containing 10 mL LB medium (10 g Bacto-tryprone, 5 gBacto-yeast extract, and 10 g aqueous sodium chloride per liter; the pHwas adjusted to 7.5 and incubated at 32° C., overnight).

A small portion of the overnight culture was placed on LB-agar (LBmedium with 15 g/L Bacto-agar) plates containing 12.5 μg/mL tetracyclinein a manner so as to obtain a single colony isolate of E. coli K12L507/pHP10D. The single colony obtained was inoculated into 10 mL of LBmedium containing 12.5 μg/mL tetracycline and incubated overnight at 32°C. with vigorous shaking. The 10 mL overnight culture was inoculatedinto LB medium containing 12.5 μg/mL tetracycline and incubated at 32°C. with vigorous shaking until the culture reached mid-log phase.

B. Culture of E. coli K12 L507/pHGAG

Lyophils of E. coli K12 L507/pHGAG were obtained from the NRRL under theaccession number NRRL B-18561 (deposited Nov. 14, 1989). A purifiedcolony of E. coli. K 12 L507/pHGAG was isolated, and used as an inoculumfor a culture which was grown to mid-log phase in substantial accordancewith the teaching of Step A, above, for E. Coli K12 L507/pHP10D.

C. Preparation of Protease Fraction

A culture of E. coli K12 L507/pHP10D was grown to mid-log phase at 32°C. in LB media containing 12.5 μg/mL tetracycline. The cultivationtemperature was quickly elevated to 40° C. to induce gene expression,and the cells were allowed to grow for 2.5 hours at this temperaturebefore the culture was quickly chilled on ice. The cells werecentrifuged and the cell pellet was resuspended in 20 mL 50 mmol MESbuffer (pH 6.0) containing 1 mmol EDTA, 1 mmol DTT, 1 mmol PMSF and 10%glycerol ("Buffer A"). Cells were lysed by sonication using a FischerModel 300 Dismembrator and a microtip probe. Following centrifugation at27,000 × g, the supernatant was diluted to a total volume of 60 mL withBuffer A and loaded onto a 2.0×19 cm QAE-Sepharose column (1 mL/min, 4°C.), that had been equilibrated in Buffer A. The column was washedisocratically for 180 min and then eluted with a gradient eluent of0-1.0M aqueous sodium chloride in Buffer A over 120 min. Enzymaticactivity was measured by HPLC using the synthetic peptide SQNYP! V asdescribed in Hargolin et al., Biochem. Biophys. Res. Commun., 167,554-560 (1990); the production of the pl peptide (SQNY) was measured.

The active fractions were combined, made 1.2M in ammonium sulfate, andapplied to a 2.0×18 cm hexyl agarose column that had been equilibratedin Buffer A containing 1.2M ammonium sulfate. The sample was loaded at aflow rate of 1 mL/min at 4° C., washed with the equilibration buffer for240 min (1 mL/min) and then eluted using a reverse linear gradient of1.2-0M ammonium sulfate in Buffer A for 120 min at the same flow rate.The column was then washed isocratically in Buffer A for 120 min.

The active fractions were combined, concentrated to 10 mL using anAmicon stirred cell with a YH-10 membrane and then applied to a HonoScation exchange column (1.0×10 cm) that had been equilibrated in BufferA. The sample was loaded at a flow rate of 1 mL/min at 25° C. Afterwashing isocratically for 30 min, the protease was eluted using a lineargradient of 0≧0.45M aqueous sodium chloride in Buffer A over 40 min..The column was washed isocratically in Buffer A containing 0.45M aqueoussodium chloride for 30 min.

The active fractions were combined and concentrated to 200 μL using anAmicon stirred cell and a YM-10 membrane and then the protease wasapplied to a Superose 6 size exclusion column equilibrated in Buffer Acontaining 0.1M aqueous sodium chloride. The column was washedisocratically in this buffer at a flow rate of 0.5 mL/min, followingwhich the HIV protease was eluted as a single peak.

QAE-Sepharose, and hexyl agarose were purchased from Sigma ChemicalCompany. Superose 6 and MonoS were were purchased from Pharmacia.Buffers and reagents were obtained from Sigma.

D. Preparation of Gag Fraction

In an analogous manner, a culture of E. coli K12 507/pHGAG was grown tomid-log phase at 32° C. then shifted to 40° C. for about 4 to 5 hours.The culture was chilled on ice and centrifuged, then the pellet wasresuspended in 8 mL lysis buffer containing 5 mg/mL lysozyme. Lysisbuffer was comprised of 50 mM Tris-HCl (pH 7.8), 5 mM EDTA, 1 mM DTT,100 mM NaCl, 1 μg/mL E64 and 2 μg/mL aprotinin. The culture wasincubated about 30 to 60 minutes at 4° C., then briefly sonicated in aBranson® Cell Disrupter at 60% power, for three 20 second bursts withchilling between each burst. The culture was then centrifuged at 15,000×g. The supernatant, which contains the unprocessed gag protein, waspartially purified by size exclusion chromatography on a Sephadex G-50column and stored at -20° C. in 50% glycerol and lysis buffer.

II. Preparation of Substrate: N.sup.α-Biotin-Gly-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(N.sup.ε -FITC)-OH A.Preparation of N.sup.α-Biotin-Gly-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys-OH

The protected peptide-resin N.sup.α-Boc-Gly-Ser-Gly-Ser-Gln-Asn-Tyr(BrZ)-Pro-Ile-Val-Gly-Lys(2-ClZ)-OCH₂-PAM-resin was synthesized on an Advanced Chemtech Model 200 peptidesynthesizer at 1.5 mmol scale using the standard double-couple protocol.The amino terminal Boc group was removed with 50% trifluoroacetic acidin methylene chloride and the resulting resin neutralized with 5%di(isopropyl)ethylamine (DIEA) in methylene chloride Then, 1.1 g (4.5mmol) of biotin in 20 mL of dimethylsulfoxide was added to the peptideresin, followed by 4.5 mmol of dicyclohexylcarbodiimide (DCC) in 9 mL ofmethylene chloride. The resulting reaction mixture was diluted to 40 mLtotal volume using 11 mL methylene chloride, and then allowed to reactfor approximately 5 hours. The reaction solution was concentrated, theresin washed sequentially with dimethyl sulfoxide, dimethylformamide andmethylene chloride and then neutralized with 5% DIEA in methylenechloride. This reaction was repeated twice, with the reaction time beingextended to 12 hours per reaction. Ninhydrin analysis of the resinindicated complete reaction of the biotin with the glycine amine group.The final peptide resin was washed extensively with dimethylformamideand methylene chloride and dried to provide 4.3 g (98%).

B. Deprotection

The peptide was deprotected and cleaved from the resin using 50 mL of ahydrofluoric acid/m-cresol solution, 0° C., 1 hour. After removal of thehydrofluoric acid by vacuum distillation, the m-cresol was extractedfrom the reaction mixture using 100 mL diethylether. The peptide wasthen solubilized in 50% aqueous acetic acid, frozen and lyophilized toprovide 2.14 g.

C. Purification

The crude N.sup.α -Biotin-Gly-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys-OH wasdissolved in 200 mL of a 5% acetonitrile (aqueous) solution containing0.1% trifluoroacetic acid and then filtered through a 0.22 micronfilter. The resulting solution was applied to a 2.2×25 cmo reverse phasecolumn of octadecyl-silica (Vydac C-18) which had been equilibrated withthe same buffer. The peptide was eluted using an 855 minute lineargradient of 7.5 to 25% acetonitrile, at 2 mL/minute, with collection offractions. These fractions were analyzed using Analytical HPLC wasperformed on a 4.6x250 mm Vydac C-18 column using similar bufferconditions. The fractions containing the desired material were combined,frozen and lyophilized to provide 1.206 g (62%).

Amino acid analysis of the isolated N.sup.α-Biotin-Gly-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gly-Lys-OH gave the followingratios: Asn 1.1; Ser 0.96; Gln 1.1; Pro 1.1; Gly 2.1; Val 0.80; Ile0.78; Tyr 1.1; Lys 1.1; in agreement with theory. Fast-atom bombardmentmass spectrometry gave a molecular ion mass peak of 1288, in agreementwith theory.

D. Labeling

The purified peptide was labeled with a fluorescent marker at theC-terminal end for use in the Pandex assay. N.sup.α-Biotin-Gly-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gly-Lys-OH (1.206 g, 0.936 mmol)was dissolved in 100 mL of 0.1M sodium borate, pH 9.5. Then, a solutionof 3 g (7.7 mmol) of fluorescein isothiocyanate in 15 mL dimethylsulfoxide was added to the reaction mixture in 10 equal portions overtwo hours. The resulting mixture was allowed to react for one hour afterthe final addition. The solution was adjusted to pH 3 using 5Nhydrochloric acid, resulting in the formation of a precipitate which wasremoved by centrifugation.

The peptide solution was then adjusted to pH 7.8 using 5N sodiumhydroxide and then diluted to 200 mL total volume by the addition of 0.1M ammonium acetate, pH 7.5. The resulting solution was then filteredthrough a 0.22 micron filter and loaded onto a 2.2×25 cm column of VydacC-18 which had been equilibrated with of 5% acetonitrile in 0.1Mammonium acetate (pH 7.5). The peptide was eluted from the column usingan 855 minute linear gradient of 5-25% acetonitrile, at 2 mL/minute,with collection of fractions. Analytical HPLC was used to analyze thefractions. The fractions containing the desired product were thencombined, frozen and lyophilized to provide 190.2 mg (12%).

Amino acid analysis of the purified peptide gave the following: Asn 1.1;Ser 1.0; Gln 1.1: Pro 1.1; Gly 2.1; Val 0.8; Ile 0.8; Tyr 1.1; Lys 1.0;in agreement with theory. Fast-atom bombardment mass spectrometry gave amolecular ion mass peak of 1678, in agreement with theory.

E. Fluorescence HIV-1 Protease Inhibitor Assay

The following buffers and solutions are used in the Fluorescence HIV-1Protease Inhibitor Assay:

MES-ALB Buffer:

0.05M 4-morpholineethane sulfonic acid, pH 5.5

0.02M NaCl

0.002M EDTA

0.001M DTT

1.0 mg/mL BSA

TBSA Buffer:

0.02M TRIS

0.15M NaCl

1.0 mg/mL BSA

Avidin Coated Beads Solution:

0.1% solution of Fluoricon Avidin Assay Particles (Avidin conjugated tosolid polystyrene beads, 0.6-0.8 microns in diameter in TBSA Buffer

Enzyme Solution:

27 IU/mL of purified HIV-1 protease in MES-ALB buffer (1 IU equals theamount of enzyme required to hydrolyze 1 μmole of substrate per minuteat 37° C.

To each well of a round bottom, 96-well plate is added 20 μL of theEnzyme Solution followed by 10 μL of the compound to be evaluated in a20% aqueous dimethylsulfoxide solution. Purified HIV-1 protease wasobtained as described above. The resulting solution is incubated for onehour at room temperature and then 20 μL of a solution containing thesubstrate, N.sup.α-Biotin-Gly-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(N.sup.ε -FITC)-OH, inHES-ALB buffer (1.5 μl/ml) is added to each well. The solutions are thenincubated for 16 hours at room temperature and then each well is dilutedwith 150 μL of MES-ALB buffer.

To each well of a second round bottom, 96-well Pandex plate is added 25μL of the Avidin Coated Beads Solution. Then, to each well is added 25μL of the diluted incubation solutions, prepared above. The solutionsare mixed thoroughly and the plates are loaded into a Pandex® machine,washed, evacuated and read. Sample detection was performed by excitationat 485 nm, reading the resulting epifluorescence at 535 nm.

The IC₅₀ results obtained in the Fluorescence Assay for the compounds ofthe present invention are set forth below in Table 1. All values havebeen normalized a positive control which is [1S-(1R*, 4R*,5S*)]-N-(1-(2-amino-2-oxoethyl)-2-oxo-3-aza-4-phenylmethyl-5-hydroxy-6-(2-(1-t-butylamino-l-oxomethyl)phenyl)hexyl)-2-quinolinylcarboxamide.

                  TABLE 1                                                         ______________________________________                                        Inhibitory Activity of Formula I Compounds                                                   Fluorescence                                                                  Assay IC.sub.50                                                Example No.    in ng/mL                                                       ______________________________________                                        Control        1.0                                                            1              0.72                                                           2              11.2                                                           ______________________________________                                    

We claim:
 1. A compound of formula I ##STR18## wherein: R is formyl orC₂ -C₆ alkanoyl;R¹ is naphthyl; q is 0; R² is --CH₂ -C(O)NH₂, or--CH(CH₃)₂ ; X is ##STR19## Y is phenyl; R^(3a) is --C(O)-NR⁴ R⁴ whereR⁴ is independently and at each occurrence hydrogen or C₁ -C₆ alkyl; ora pharmaceutically acceptable salt thereof.
 2. A compound according toclaim 1 wherein:R is ethanoyl; R¹ is naphth-2-yl; R² is --CH₂ --C(O)NH₂; and R^(3a) is --C(O)--NH(t-butyl);or a pharmaceutically acceptablesalt thereof.
 3. A compound according to claim 2 which is[2'R-(2'R*,3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3'-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl(]nonylbenzamide;or a pharmaceutically acceptable salt thereof.
 4. Apharmaceutical formulation comprising one or more pharmaceuticallyacceptable carriers, diluents or excipients and a compound of claim 1.5. A pharmaceutical formulation according to claim 4 where the compoundis one wherein:R is ethanoyl; R¹ is naphth-2-yl; R² is --CH₂ --C(O)NH₂ ;and R^(3a) is --C(O)--NH(t-butyl);or a pharmaceutically acceptable saltthereof.
 6. A pharmaceutical formulation according to claim 5 where thecompound is[2'R-(2'R*,3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-3'-naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylbenzamide;or a pharmaceutically acceptable salt thereof.
 7. A method ofinhibiting HIV replication comprising administering to an HIV infectedcell or a cell susceptible to HIV infection an effective amount of acompound of claim
 2. 8. A method according to claim 7 where the compoundis one wherein:R is ethanoyl; R₁ is naphth-2-yl; R² is --CH₂ --C(O)NH₂ ;and R^(3a) is --C(O)--NH(t-butyl);or a pharmaceutically acceptable saltthereof.
 9. A method to claim 8 where the compound is[2'R-(2'R-(2'R*,3'R*,6'S*)]-N(t-butyl)-2-[2'-hydroxy-2'Naphth-2-ylthiomethyl-4',7'-diaza-5',8'-dioxo-6'-(2"-amino-2"-oxoethyl)]nonylbenzamide;or a pharmaceuticaly acceptable salt thereof.